Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of Munc18

Munc18a, a mammalian neuronal homologue of Saccharomyces cerevisiae Sec1p p rotein, is essential for secretion, likely as a result of its high affinity interaction with the target SNARE protein syntaxin 1a (where SNARE is deri ved from SNAP receptor (the soluble N-ethylmaleimide-sensitive fusion prote in)). However, this interaction inhibits vesicle SNARE interactions with sy ntaxin that are required for secretory vesicles to achieve competency for m embrane fusion, As such, regulation of the interaction between Munc18a and syntaxin la may provide an important mechanism controlling secretory respon siveness. Cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division kinases, co-purifies with Munc18a from rat brain, interacts d irectly with Munc18a in vitro, and utilizes Munc18a as a substrate for phos phorylation, We have now demonstrated that Cdk5 is capable of phosphorylati ng Munc18a in vitro within a preformed Munc18a syntaxin la heterodimer comp lex and that this results in the disassembly of the complex, Using site-dir ected mutagenesis, the Cdk5 phosphorylation site on Munc18a was identified as Thr(574). Stimulation of secretion from neuroendocrine cells produced a corresponding rapid translocation of cytosolic Cdk5 to a particulate fracti on and an increase of Cdk5 kinase activity. Inhibition of Cdk5 with olomouc ine decreased evoked norepinephrine secretion from chromaffin cells, an eff ect not observed with the inactive analogue iso-olomoucine, The effects of olomoucine were independent of calcium influx as evidenced by secretary inh ibition in permeabilized chromaffin cells and in cells under whole-cell vol tage clamp. Furthermore, transfection and expression in chromaffin cells of a neural specific Cdk5 activator, p25, led to a strong increase in nicotin ic agonist-induced secretory responses. Our data suggest a model whereby Cd k5 acts to regulate Munc18a interaction with syntaxin la and thereby modula tes the level of vesicle SNARE interaction with syntaxin 1a and secretory r esponsiveness.